U.S. patent application number 13/272942 was filed with the patent office on 2012-05-03 for cell sorting apparatus, cell sorting chip, and cell sorting method.
Invention is credited to Yoichi Katsumoto.
Application Number | 20120107860 13/272942 |
Document ID | / |
Family ID | 45997180 |
Filed Date | 2012-05-03 |
United States Patent
Application |
20120107860 |
Kind Code |
A1 |
Katsumoto; Yoichi |
May 3, 2012 |
CELL SORTING APPARATUS, CELL SORTING CHIP, AND CELL SORTING
METHOD
Abstract
Disclosed herein is a cell sorting apparatus, including: a
branch portion branching a flow path in which a fluid containing
therein cells flows into a first branch flow path, and a second
branch flow path; a coupling portion coupling the first branch flow
path and the second branch flow path to each other; and a
flowing-out portion causing liquids flowing in the first branch
flow path and the second branch flow path coupled to each other by
the coupling portion, respectively, to flow out to an outside.
Inventors: |
Katsumoto; Yoichi; (Tokyo,
JP) |
Family ID: |
45997180 |
Appl. No.: |
13/272942 |
Filed: |
October 13, 2011 |
Current U.S.
Class: |
435/29 ;
435/287.1 |
Current CPC
Class: |
B03C 5/026 20130101;
C12M 47/04 20130101; G01N 15/1056 20130101; B03C 5/005 20130101;
B01L 3/502761 20130101; G01N 2015/1081 20130101; B03C 2201/26
20130101 |
Class at
Publication: |
435/29 ;
435/287.1 |
International
Class: |
C12Q 1/02 20060101
C12Q001/02; C12M 1/34 20060101 C12M001/34 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 29, 2010 |
JP |
P2010-244004 |
Claims
1. A cell sorting apparatus, comprising: a branch portion branching
a flow path in which a fluid containing therein cells flows into a
first branch flow path, and a second branch flow path; a coupling
portion coupling said first branch flow path and said second branch
flow path to each other; and a flowing-out portion causing liquids
flowing in said first branch flow path and said second branch flow
path coupled to each other by said coupling portion, respectively,
to flow out to an outside.
2. The cell sorting apparatus according to claim 1, further
comprising: a cell holding portion provided in said first branch
flow path and holding the cells, wherein a length of said cell
holding portion, and a ratio between an average cross-sectional
area of said cell holding portion and an average cross-sectional
area of said first branch flow path are set in such a way that a
distance by which the cells settle out while the cells pass through
said cell holding portion becomes larger than a height of said
first branch flow path.
3. A cell sorting chip, comprising: a substrate; a flow path which
is provided in said substrate and in which a fluid containing
therein cells flows; a first branch flow path and a second branch
flow path provided in said substrate and branching at said flow
path; a cell holding portion provided in said first branch flow
path and holding the cells contained in the fluid flowing in said
first branch flow path; and a flowing-out portion coupling said
first branch flow path and said second branch flow path to each
other and causing liquids flowing in said first branch flow path
and said second branch flow path to flow out to an outside.
4. The cell sorting chip according to claim 3, wherein said cell
holding portion has a film-like portion adapted to be dug from an
outside.
5. A cell sorting method, comprising: branching a flow path in
which a fluid containing therein cells flows into a first branch
flow path and a second branch flow path; coupling said first branch
flow path and said second branch flow path to each other; and
causing liquids flowing in said first branch flow path and said
second branch flow path coupled to each other, respectively, to
flow out to an outside.
Description
CROSS REFERENCES TO RELATED APPLICATIONS
[0001] The present application claims priority to Japanese Patent
Application No. 2010-244004 filed on Oct. 29, 2010, the disclosure
of which is incorporated herein by reference.
BACKGROUND
[0002] The present disclosure relates to a cell sorting apparatus,
a cell sorting chip, and a cell sorting method for sorting desired
cells.
[0003] A fluorescence flow cytometer or a cell sorter is known as
an apparatus for sorting cells. With such a sorting apparatus,
cells are trapped in an air-liquid interface in an outlet port
under a suitable vibration condition (in general, a flow rate at an
outlet port is several meters/second, and a frequency is several
tens of kilohertz) by a circumferential fluid. At the same time,
the electric charges are given to each of the cells. Each of the
cells flies as a droplet in an air to which a static electric field
is applied in a direction corresponding to a quantity of electric
charges. Finally, the cells are sorted into sorting containers
provided outside a flow path.
[0004] This technique is useful when the flow rate is relatively
high as with the case described above. However, in the flow
cytometer or dielectric cytometer showing the low flow rate, it is
difficult for this technique to fulfill the conditions of the
droplet and the discharge. For this reason, if anything, it is
preferable that the sorting operation is carried out within the
flow path having a branch provided therein, and the cells are held
in a preceding stage.
[0005] With regard to a method of introducing the cells after the
branching into the containers, for example, as described in
JP-T-2003-507739 (refer to FIG. 38 and the like), there is known a
method of drawing a flow containing therein cells to the outside of
an apparatus through a pipe.
[0006] However, when the flow containing therein the cells is drawn
to the outside of the apparatus through the pipe, in general, a
liquid sending path becomes long, it takes a lot of time for the
flow to reach the container, and a consumption of an amount of
liquid is also large.
SUMMARY
[0007] In order to cope with such a situation, the inventors of
this technology proposed a cell sorting method of carrying out
intra-flow path sorting for cells based on some sort of cell
sorting information. In an apparatus realizing this method, a
sorting driving force applying portion for applying a driving force
to cells in order to sort the cells by changing a path for the
cells based on the cell sorting information is provided within a
flow path. In addition, this apparatus is provided with two branch
flow paths and liquid staying portions. In this case, the two
branch flow paths are provided downstream with respect to the
sorting driving force applying portion. Also, in the liquid staying
portions, the fluids flowing through the two branch flow paths,
respectively, are made to stay.
[0008] However, in such an apparatus, it is possible that a water
head difference or the like in the liquid staying portions are
readily generated, and this influence is propagated as a pressure
change up to the sorting driving force applying portion through the
branch flow paths, thereby impeding the precise sorting of the
cells by the sorting driving force applying portion.
[0009] The present technology has been made in order to solve the
problems described above, and it is therefore desirable to provide
a cell sorting apparatus, a cell sorting chip, and a cell sorting
method which can precisely sort cells without generating a pressure
difference between branch flow paths.
[0010] According to an embodiment of the present disclosure, there
is provided a cell sorting apparatus including: a branch portion
branching a flow path in which a fluid containing therein cells
flows into a first branch flow path, and a second branch flow path;
a coupling portion coupling the first branch flow path and the
second branch flow path to each other; and a flowing-out portion
causing liquids flowing in the first branch flow path and the
second branch flow path coupled to each other by the coupling
portion, respectively, to flow out to an outside.
[0011] In the embodiment of the present disclosure, the first
branch flow path and the second branch flow path which branch once
are coupled to each other again by the coupling portion. Therefore,
when the fluids flowing in the first branch flow path and the
second branch flow path, respectively, are caused to flow out from
the flowing-out portion to the outside, no pressure difference is
generated between the first branch flow path and the second branch
flow path. Thus, the cells can be precisely sorted.
[0012] Preferably, the cell sorting apparatus may further include a
cell holding portion provided in the first branch flow path and
holding the cells, in which a length of the cell holding portion,
and a ratio between an average cross-sectional area of the cell
holding portion and an average cross-sectional area of the first
branch flow path are set in such a way that a distance by which the
cells settle out while the cells pass through the cell holding
portion becomes larger than a height of the first branch flow
path.
[0013] The setting is made in such a way, whereby the desired cells
sorted are held by the cell holding portion without flowing into
the flowing-out portion. Therefore, the cells sorted can be
reliably taken out to the outside from the cell holding
portion.
[0014] According to another embodiment of the present disclosure,
there is provided a cell sorting chip including: a substrate; a
flow path which is provided in said substrate and in which a fluid
containing therein cells flows; a first branch flow path and a
second branch flow path provided in the substrate and branching at
the flow path; a cell holding portion provided in the first branch
flow path and holding the cells contained in the fluid flowing in
the first branch flow path; and a flowing-out portion coupling the
first branch flow path and the second branch flow path to each
other and causing liquids flowing in the first branch flow path and
the second branch flow path, respectively, to flow out to an
outside.
[0015] In the embodiment of the present disclosure, the first
branch flow path and the second branch flow path which branch once
are coupled to each other again. Therefore, when the fluids flowing
in the first branch flow path and the second branch flow path,
respectively, are caused to flow out from the flowing-out portion
to the outside, no pressure difference is generated between the
first branch flow path and the second branch flow path. Thus, the
cells can be precisely sorted. Also, the cells sorted can be taken
out from the cell holding portion to the outside.
[0016] Preferably, said cell holding portion may have a film-like
portion adapted to be dug from an outside.
[0017] The cell holding portion has the film-like portion adapted
to be dug from the outside. Therefore, a pipette, for example, is
put in the cell holding portion through the film-like portion,
which results in that the cells held in the cell holding portion
can be simply taken out to the outside by using the pipette or the
like.
[0018] According to still another embodiment of the present
disclosure, there is provided a cell sorting method including:
branching a flow path in which a fluid containing therein cells
flows into a first branch flow path and a second branch flow path;
coupling said first branch flow path and the second branch flow
path to each other; and causing liquids flowing in the first branch
flow path and the second branch flow path coupled to each other,
respectively, to flow out to an outside.
[0019] In the embodiment of the present disclosure, the first
branch flow path and the second branch flow path which branch once
are coupled to each other again. Therefore, when the fluids flowing
in the first branch flow path and the second branch flow path,
respectively, are caused to flow out from the flowing-out portion
to the outside, no pressure difference is generated between the
first branch flow path and the second branch flow path. Thus, the
cells can be precisely sorted.
[0020] As set forth hereinabove, according to the present
disclosure, since the first branch flow path and the second branch
flow path which branch once are coupled to each other again, no
pressure difference is generated between the first branch flow path
and the second branch flow path. Thus, the cells can be precisely
sorted.
[0021] Additional features and advantages are described herein, and
will be apparent from the following Detailed Description and the
figures.
BRIEF DESCRIPTION OF THE FIGURES
[0022] FIG. 1 is a conceptual view of a cell function
analyzing/sorting system according to an embodiment of the present
disclosure.
[0023] FIG. 2 is a perspective view showing a structure of a cell
sorting chip which can be applied to the cell function
analyzing/sorting system shown in FIG. 1.
[0024] FIG. 3 is a top plan view showing a structure of a sorting
portion (a sorting signal is held in an OFF state) shown in FIG.
2.
[0025] FIG. 4 is a cross sectional view taken on line A-A of FIG.
3.
[0026] FIG. 5 is a top plan view showing the structure of the
sorting portion (the sorting signal is held in an ON state) shown
in FIG. 2.
[0027] FIG. 6 is a top plan view showing a structure of a cell
holding portion and a flowing-out portion both shown in FIG. 2.
[0028] FIG. 7 is a cross sectional view taken on line A-A of FIG.
6.
[0029] FIG. 8 is a schematic cross sectional view of the cell
holding portion shown in FIG. 6.
[0030] FIG. 9 is a view explaining an operation and effects
relating to the present disclosure.
DETAILED DESCRIPTION
[0031] Embodiments of the present application will be described
below in detail with reference to the drawings.
[0032] (Outline of Cell Function Analyzing/Sorting System)
[0033] FIG. 1 is a conceptual view of a cell function
analyzing/sorting system according to an embodiment of the present
disclosure.
[0034] As shown in FIG. 1, the cell function analyzing/sorting
system 1 is provided with a putting-in portion 3, a measuring
portion 4, a sorting portion 5, cell holding portions 6 and 7, and
a flowing-out portion 10 from an upstream side of a micro-flow path
(hereinafter referred simply to as "a flow path") 2 along the flow
path. The sorting portion 5 includes an electric field applying
portion 8 and a branch portion 9.
[0035] Reference symbol C designates a cell. A liquid containing
therein the cells C sampled is put in a pressure container (not
shown), for example, using a pump or the like.
[0036] The liquid put in from the putting-in portion 3 flows in the
flow path 2. The flow path 2 branches into a branch flow path 2a
and a branch flow path 2b at the branch portion 9. The branch flow
path 2a and the branch flow path 2b branching once are coupled
again in a flowing-out portion 10. Although the flowing-out portion
10 has a function as such a coupling portion, the coupling portion
may be provided separately from the flowing-out portion 10. The
cell holding portion 6 is provided in the middle of the branch flow
path 2a and the cell holding portion 7 is provided in the middle of
the branch flow path 2b.
[0037] The measuring portion 4 measures complex permittivities of
the individual cells C flowing in the flow path 2 over multipoint
frequencies (three points or more in frequency, typically, in the
range of about 10 to about 20 points in frequency) in the frequency
range (for example, in the range of 0.1 to 50 MHz) in which a
dielectric relaxation phenomenon of the cells C occurs. The
measuring portion 4 determines whether or not the cells C are cells
to be sorted in accordance with the complex permittivities of the
cells C thus measured. When it is determined that the cells C are
cells to be sorted, the measuring portion 4 outputs a sorting
signal. Instead of providing the measuring portion 4, a portion
corresponding thereto, for example, may be composed of a signal
detecting portion composed of a pair of electrodes, and a cell
function analyzing portion for analyzing functions of the cells C
in accordance with a signal detected.
[0038] The sorting portion 5 sorts the desired cells C of plural
kinds of cells C put in from the putting-in portion 3 into the cell
holding portion 6, and sorts the cells other than the desired cells
C into the cell holding portion 7.
[0039] The electric field applying portion 8 can apply an electric
field having a gradient in a direction different from an X
direction in which the fluid flows, for example, in a Y direction
perpendicular to the X direction. For example, the electric field
applying portion 8 does not apply the electric field when the
sorting signal is not inputted, but the electric field applying
portion 8 applies the electric field when the sorting signal is
inputted.
[0040] The branch portion 9 branches the cells C to which no
electric field is applied in the electric field applying portion 8
in such a way that the cells C flow in the cell holding portion 7.
Also, the branch portion 9 branches the cells C to which the
electric field is applied in the electric field applying portion 8
in such a way that the cells C flow in the cell holding portion
6.
[0041] The liquids which have passed through the cell holding
portions 6 and 7, respectively, are discharged from the flowing-out
portion 10 to the outside through the pressure container, for
example, using the pump. A pressure difference is generated in the
flow path 2 due to application of a pressure in the putting-in
portion 3, and reduction of a pressure in the flowing-out portion
10.
[0042] In the cell function analyzing/sorting system 1, the
electric field is applied to the cells based on ON/OFF or amplitude
modulation of the electric field in accordance with the sorting
signal which is previously outputted either from a measuring
portion or from a measured value analyzing portion by using some
sort of another technique. Thus, even in the case of a cell group
having a dispersion in cell diameter or physical property of the
cells C, only the cells C as an object of the sorting are sorted
based on a sufficient dielectrophoretic force.
[0043] (Chip for Analyzing/Sorting Cell Functions)
[0044] FIG. 2 is a perspective view showing a structure of a cell
sorting chip which is applied to the cell function
analyzing/sorting system shown in FIG. 1.
[0045] As shown in FIG. 2, a chip 11 includes a substrate 12 and a
sheet-like member 13 made of a polymer film or the like. The
substrate 12 is provided with the flow path 2, a liquid putting-in
portion 3a as the putting-in portion 3, the branch portion 9, the
cell holding portions 6 and 7, and the flowing-out portion 10.
Trenches or the like are formed in a surface of the substrate 12,
and surfaces thereof are covered with the sheet-like member 13,
thereby structuring these constituent elements. The cell putting-in
portion 3b in which the liquid containing therein the cells C is
put is structured by providing a fine hole in the sheet-like member
13. When the liquid containing therein the cells C dropped on the
cell putting-in portion 3b with a pipette, the liquid containing
therein the cells C flows downstream with respect to the flow path
2 so as to be dragged in the liquid flowing in the flow path 2
through the fine hole. Because of the fine hole, plural cells C do
not collectively flow into the flow path 2, but plural cells C flow
into the flow path 2 one by one.
[0046] A pair of signal detecting electrodes 4a and 4b are provided
so as to hold the fine hole between the signal detecting electrodes
4a and 4b. One signal detecting electrode 4a is provided on a front
surface of the sheet-like member 13, and the other signal detecting
electrode 4b is provided on a back surface of the sheet-like member
13. An electrode pair which will be described later and which
composes the electric field applying portion 8 is also formed on
the back surface of the sheet-like member 13.
[0047] Upper portions of the cell holding portions 6 and 7 are
covered with the sheet-like member 13 as a film-like portion which
can be dug from the outside. However, the pipette is stung to the
sheet-like member 13, thereby making it possible to take out the
cells C through the pipette.
[0048] Electrode pads 14 take out a signal detected by the signal
detecting electrodes 4a and 4b to the outside. The signal thus
taken out, for example, is sent to a cell function analyzing
portion (not shown). The sorting signal outputted from the cell
function analyzing portion is inputted to electrode pads 15. The
sorting signal thus inputted is then sent to the electrode pair
composing the electric field applying portion 8.
[0049] Through holes 26 are positioning holes when the chip 11 is
mounted to an apparatus main body having the cell function
analyzing portion and the like.
[0050] (Structure of Sorting Portion)
[0051] FIG. 3 is a top plan view showing a structure of the sorting
portion 5 shown in FIG. 2, and FIG. 4 is a cross sectional view
taken on line A-A of FIG. 3.
[0052] As shown in FIGS. 3 and 4, the sorting portion 5 includes
the electric field applying portion 8 and the branch portion 9.
[0053] The electric field applying portion 8 includes electrodes 16
and 17 provided in predetermined positions of the flow path 2,
respectively. The electrodes 16 and 17 are disposed so as to hold
the flow path 2 between the electrodes 16 and 17 in a direction
different from a direction (X direction) of the fluid flowing in
the flow path 2, for example, in a Y direction and so as to face
each other. The electrodes 16 and 17 are provided on a back surface
(an upper surface within the flow path 2) of the sheet-like member
13. The electrode 16, for example, is an electrode to which a
signal is applied and is structured in such a way that a large
number of electrode fingers 16a protrude toward the electrode 17.
The electrode 17, for example, is a common electrode and is
structured so as not to have irregularities for the electrode 16.
In the following description, a combination of one electrode finger
16a and the electrode 17 is referred to as an electrode pair 18.
The electrode pair 18 is structured in such a way, whereby when the
signal is applied across the electrodes 16 and 17, the electric
fields which have gradients in the Y direction are applied across
the electrode pairs 18, respectively.
[0054] The branch portion 9 branches the cells C whose flow
directions are changed by the dielectrophoretic force generated by
application of the electric field by the electric field applying
portion 8 into the branch flow path 2a and the branch flow path 2b
in a predetermined position located downstream with respect to the
electric field applying portion 8 within the flow path 2. The flow
path 2 branches into a Y letter shape, thereby structuring the
branch portion 9. One branch portion extends toward the cell
holding portion 6 through the branch flow path 2a, and the other
branch portion extends toward the cell holding portion 7 through
the branch flow path 2b. For example, in the putting-in portion 3,
the cells C are put in a biased position on the cell holding
portion 7 side. With regard to the cells C put in the biased
position on the cell holding portion 7 side in such a way, when the
cells C which are not as an object of the sorting pass through the
electric field applying portion 8, no electric field is applied to
any of the cells in the electric field applying portion 8
(non-active). Thus, as shown in FIG. 3, the cells C concerned flow
in the flow path 2 on the biased position side to flow in the cell
holding portion 7 as they are. On the other hand, when the cells C
which are as an object of the sorting pass through the electric
field applying portion 8, the electric field is applied to the
cells concerned in the electric field applying portion 8 (active),
and thus the dielectrophoretic force is applied to the cells C. As
a result, as shown in FIG. 5, the flow direction of the cells C is
changed to the cell holding portion 6 side, and thus the cells C as
the object of the sorting are branched to the cell holding portion
6 side by the branch portion 9.
[0055] In the electric field applying portion 8 structured in such
a way, the electric fields which have the gradients in the Y
direction in the electrode pairs 18, respectively, are applied.
Therefore, the cells C passing through the electric field applying
portion 8 can be gradually changed in the path thereof to branch to
the cell holding portion 6 side.
[0056] (Structures of Cell Holding Portion and Flowing-out
Portion)
[0057] FIG. 6 is a top plan view showing structures of the cell
holding portion and the flowing-out portion, and FIG. 7 is a cross
sectional view taken on line A-A of FIG. 6.
[0058] As shown in FIGS. 6 and 7, the cell holding portion 6 is
provided in the middle of the branch flow path 2a, and the cell
holding portion 7 is provided in the middle of the branch flow path
2b. A termination of the branch flow path 2a and a termination of
the branch flow path 2b are coupled to each other again at the
flowing-out portion 10. Therefore, the liquid flowing in the branch
flow path 2a passes through the cell holding portion 6 so as to
flow into the flowing-out portion 10, and the liquid flowing in the
branch flow path 2b passes through the cell holding portion 7 so as
to flow into the flowing-out portion 10.
[0059] Each of the cell holding portions 6 and 7 operates as a flow
path abruptly expanding portion. Thus, the cells C do not flow into
the flowing-out portion 10, but are held in the cell holding
portions 6 and 7.
[0060] For example, each of the cell holding portions 6 and 7 is
composed of a bottomed hole having a cylindrical shape which is
sufficiently deeper in depth than each of the branch flow paths 2a
and 2b, and is sufficiently larger in diameter than a width of each
of the branch flow paths 2a and 2b.
[0061] As described above, in each of the cell holding portions 6
and 7, an average cross-sectional area of a surface vertical to the
flow direction (X direction) of the liquid is sufficiently larger
than a cross-sectional area of a surface of each of the branch flow
paths 2a and 2b vertical to the flow direction. Therefore, a
settling-out velocity, v, of each of the cells C becomes larger
than a flow rate, u, of each of the cells C in the flow
direction.
[0062] The cell holding portions 6 and 7 are suitably designed,
which results in that although the fluid itself usually flows out
from the flowing-out portion 10 to the container(s) in the outside
of the apparatus, the cells C settling out in the cell holding
portions 6 and 7 stay in recirculation regions or dead water
regions generated in the cell holding portions 6 and 7,
respectively, and thus do not flow out to the flowing-out portion
10. That is to say, in order to attain this, it is only necessary
that a ratio between a length of each of the cell holding portions
6 and 7 and the average cross-sectional area of each of the cell
holding portions 6 and 7, and the average cross-sectional area of
each of the branch flow paths 2a and 2b is set in such a way that a
distance by which the cells C settle out while the cells C pass
through the cell holding portions 6 and 7 becomes larger than a
height of each of the branch flow paths 2a and 2b.
[0063] Here, as shown in FIG. 8, a time for which the cells C pass
through the cell holding portion 6, 7 is given by t, an average
flow rate of the cells C within the branch flow path 2a, 2b in a
mainstream direction (X direction) is given by u.sub.1, an average
flow rate in a height direction (Z direction (a gravity direction
is set as positive)) is given by v.sub.1, an average flow rate of
the cells C within the cell holding portion 6, 7 in the mainstream
direction (X direction) is given by u.sub.2, and an average flow
rate in the height direction (Z direction (the gravity direction is
set as positive)) is given by v.sub.2. In addition, with regard to
a surface vertical to the mainstream direction (X direction), an
average cross-sectional area of the branch flow path 2a, 2b is
given by A.sub.1, an average cross-sectional area of the cell
holding portion 6, 7 is given by A.sub.2, a flow path height within
the branch flow path 2a, 2b is given by h, and a mainstream
direction length of the cell holding portion 6, 7 is given by
L.
[0064] A settling-out velocity in the gravity direction is obtained
from the Stokes equation, more strictly, from a drag acting on a
ball in a uniform stream. When the settling-out velocity in the
gravity direction is given by v.sub.s, a relationship of
v.sub.1=v.sub.2=v.sub.s is obtained because v.sub.s does not depend
on the flow rate in the mainstream direction.
[0065] Now, a time, t, required for the cells C to pass through the
cell holding portion 6, 7 is expressed by Expression (1):
t=L/u.sub.2=(L/u.sub.1).times.(A.sub.2/A.sub.1) (1)
[0066] At this time, a distance, z.sub.s, by which the cells C
settle out for the time, t, is expressed by Expression (2):
z.sub.s=v.sub.s.times.t (2)
[0067] when z.sub.s is larger than the flow path height, h, the
cells C are trapped in a circulating flow or the dead water region
existing below the cell holding portion 6, 7, and thus stay in the
cell holding portion 6, 7 without flowing into the flowing-out
portion 10. That is to say, it is only necessary for this condition
to fulfill Conditional Expression (3):
z.sub.s=v.sub.s.times.t>h (3)
[0068] A description will be further given below by using concrete
numerical values.
[0069] As an example of the cells C, a specific gravity,
.rho..sub.cell, of a white blood cell is in the range of 1.063 to
1.085, and a typical value thereof is given by 1.07. Also, a
diameter of the cell C is given by 10 .mu.m, in a word, a radius,
r, of the cell C is given by 5 .mu.m.
[0070] A density, .rho..sub.H2O, of water as an example of a fluid
is given by 1,000 kg/cm.sup.3 at 20.degree. C., and a viscosity
coefficient, .mu., of the water is given by 0.001 PaS at 20.degree.
C.
[0071] In this case, the settling-out velocity, v.sub.s, of the
cell C in the gravity direction is given from the Stokes equation
by:
v.sub.s=2/9.times.(.rho..sub.cell-.rho..sub.H2O)gr.sub.2/.mu.=2.2
(.mu.m/s)
[0072] In addition, the settling-out velocity, v.sub.s, of the cell
C in the gravity direction is given from a general drag equation
for the ball by: v.sub.s=3.8 (.mu.m/s)
[0073] Here, when a mainstream direction length L of the cell
holding portion 6, 7 is L=5 (nm), the ratio S.sub.r
(A.sub.1:A.sub.2) between the average cross-sectional area A.sub.1
of the branch flow path 2a, 2b, and the average cross-sectional
area A.sub.2 of the cell holding portion 6, 7 in the mainstream
direction (X direction) is S.sub.r=100, and the average flow rate
u.sub.1 of the cell C within the branch flow path 2a, 2b in the
mainstream direction (X direction) is u.sub.1=10 (.mu.m/s), the
flow path height, h, within the branch flow path 2a, 2b is h =50
(.mu.m).
[0074] Also, when the time, t, for which the cell C passes through
the cell holding portion 6, 7 is t=50 (s), the distance, z.sub.s,
by which the cell C settles out for the time, t, is given by
z.sub.s=v.sub.s.times.t=3.8 (.mu.m/s).times.50 (s)=190 (.mu.m).
[0075] Since this calculation result fulfills a relationship of
z.sub.s>h, the cell C is trapped in the circulating flow or the
dead water region existing below the cell holding portion 6, 7, and
thus stay in the cell holding portion 6, 7 without flowing into the
flowing-out portion 10. In addition, even when a dispersion of the
cell diameter is set to .+-.2 (.mu.m) and the calculation is
similarly carried out with respect to the minimum diameter cell, a
relationship of z.sub.s=130 (.mu.m)>h is obtained. Thus, in the
case as well, the cell C is trapped in the circulating flow or the
dead water region existing below the cell holding portion 6, 7, and
thus stay in the cell holding portion 6, 7 without flowing into the
flowing-out portion 10.
[0076] From the foregoing, when both of the practical size of the
chip 11, and the sorting speed used in the apparatus are taken into
consideration, it is understood that an achievable design
resolution exists.
[0077] (Cell Sorting Method)
[0078] A cell sorting method according to another embodiment of the
present disclosure includes: branching the flow path 2 in which the
fluid containing therein the cells C flows into the branch flow
path 2a and the branch flow path 2b; coupling the branch flow path
2a and the branch flow path 2b to each other; and causing the
liquid flowing in the branch flow path 2a and the branch flow path
2b coupled to each other, respectively, to flow out to the
outside.
[0079] (Operation and Effects)
[0080] As shown in FIG. 9, for the purpose of carrying out the
intra-flow path sorting for the cells C based on some sort of cell
sorting information, the apparatus needs to include at least the
sorting portion 5 within the flow path 2, and the branch flow paths
2a and 2b, and the flowing-out portions 10a and 10b which are all
located downstream with respect to the sorting portion 5. In
addition, it is necessary to previously realize the stable liquid
sending.
[0081] With a method of sending the liquid within the flow path
based on the pressure difference by using the pressure container
using the pump or the like, since the fluid can be isolated from
the outside atmospheric pressure by adopting the sealed structure,
the stable liquid sending can be carried out as compared with the
liquid sending by using the pump in which the pulsation essentially
exists. In this case, with regard to the fluid flowing out from the
branch portion 9 into the flowing-out portions 10a and 10b, a
static pressure in each of the flowing-out portions 10a and 10b
needs to be stable, and the static pressures in the flowing-out
portions 10a and 10b need to be equal to each other, or needs to be
usually, strictly kept at a desired ratio. The reason for this is
because when the static pressures in the flowing-out portions 10a
and 10b are changed, the amounts of fluids which flow from the
mainstream into the branch flow paths 2a and 2b through the branch
portion 9 are changed in accordance with a ratio in difference
pressure between the static pressure in the branch portion 9, and
the static pressure in each of the flowing-out portions 10a and
10b. When the amounts of fluids are changed, the branch flow paths
2a and 2b into which the cells flow are readily changed, so that
even when how a driving force for the sorting is applied either to
the cells themselves or to the entire fluid containing therein the
cell C short of the branch portion 9, it may be impossible to send
the cells C to the desired flowing-out portion 10a, 10b no
longer.
[0082] Then, in the embodiments of the present disclosure, when the
pressure driving is carried out, the flowing-out portions are
coupled to each other, and the fluid is discharged into the
low-pressure side pressure container in the outside, which results
in that the stable liquid sending free from the pressure change
becomes possible. That is to say, according to the embodiments of
the present disclosure, no pressure difference is generated between
the branch flow paths 2a and 2b, and thus it is possible to readily
realize the stable liquid sending which is essential to the cell
sorting within the flow path 2. In addition, the cells C after the
sorting stay in the cell holding portions 6 and 7 within the chip
11, and thus it is unnecessary to specially prepare the pipe and
the container, to say nothing of causing the cells C to fly in the
air. Therefore, it is possible to readily and inexpensively realize
the contamination-free and thus it is possible to apply the present
disclosure to the regenerative medicine.
[0083] It is noted that the present disclosure is by no means
limited to the embodiments described above, and various kinds of
changes can be made within the range of the technical idea.
[0084] For example, although in the embodiments described above,
the case where the coupling portion and the flowing-out portion are
integrated with each other is exemplified, the coupling portion and
the flowing-out portion may also exist separately from each other.
For example, the two branch flow paths may be coupled to each other
once at the coupling portion to be connected to the flowing-out
portion through the common flow path.
[0085] Although in the embodiments described above, the case where
the two branch flow paths are provided is exemplified, three or
more branch flow paths may also be provided.
[0086] Although in the embodiments described above, the case where
the cell holding portion is provided in each of the branch flow
paths is shown, a structure may also be adopted such that the cell
holding portion is provided only in the branch flow path for
sorting the cells.
[0087] Although the shape, size and the like of each of the cell
holding portions are by no means limited to these in the
embodiments described, of course, each of the cell holding portions
can be embodied in various forms.
[0088] It should be understood that various changes and
modifications to the presently preferred embodiments described
herein will be apparent to those skilled in the art. Such changes
and modifications can be made without departing from the spirit and
scope and without diminishing its intended advantages. It is
therefore intended that such changes and modifications be covered
by the appended claims.
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